Mutant gamma-glutamyltransferase, and a method for producing gamma-glutamylvalylglycine or a salt thereof

ABSTRACT

A method for producing γ-Glu-Val-Gly comprising the step of reacting Val-Gly with a γ-glutamyl group donor in the presence of a γ-glutamyltransferase, a microorganism containing the enzyme, or a processed product thereof to generate γ-Glu-Val-Gly, wherein the γ-glutamyltransferase consists of a large subunit and a small subunit, and the small subunit has a specific mutation.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a continuation of and claims the benefits ofpriority to International Application No. PCT/JP2012/075908, filed Oct.5, 2012, the entire contents of which are incorporated herein byreference. International Application No. PCT/JP2012/075908 claims thebenefits of priority to Japanese Patent Application No. 2011-223058,filed Oct. 7, 2011.

TECHNICAL FIELD

The present invention relates to a method for producingγ-glutamylvalylglycine or a salt thereof, and a mutant ofγ-glutamyltransferase preferably used for the method.γ-Glutamylvalylglycine is useful in the fields of food, drug, and soforth.

BACKGROUND ART

A certain kind of peptides such as γ-glutamylvalylglycine(L-γ-glutamyl-L-valyl-glycine, henceforth referred to as“γ-Glu-Val-Gly”) have a calcium receptor agonistic activity (Patentdocument 1). Such peptides having a calcium-receptor activation actionare known to be able to impart kokumi taste to foods and drinks (Patentdocument 2), improve tastes of low fat diets, especially fat-likethickness and smoothness (Patent document 3), improve feeling of body ofsweet taste substances, and improve bitterness peculiar to sweet tastesubstances (Patent document 4).

Moreover, such peptides as described above are known to have aprophylactic or curative effect on diarrhea (Patent document 5) anddiabetes (Patent document 6), and a bicarbonate secretion promotingeffect in the alimentary tract (Patent document 7).

As described above, wide application of γ-Glu-Val-Gly in the field offood, drug, and so forth is expected.

As methods for producing tripeptides, chemical synthesis methods andenzymatic methods are generally known. As one of the chemical synthesismethods, a method of selectively γ-glutamylating a dipeptide by usingN-protected glutamic anhydride to obtain a tripeptide is known (Patentdocument 10). As the enzymatic methods, there is known, for example, amethod of reacting a dipeptide having an esterified or amidated carboxylterminus and an amino acid having free amino group (for example, anamino acid of which carboxyl group is protected) in the presence of apeptide-producing enzyme to produce a tripeptide (Patent document 8).

As an enzyme that catalyzes the reaction of transferring γ-glutamylgroup to a dipeptide, γ-glutamyl transferase (also called γ-glutamyltranspeptidase, henceforth also referred to as “GGT”) is known. It wasreported that in a reaction of Val-Gly (valylglycine) andγ-glutamyl-p-nitroanilide as a glutamine donor in the presence of thatenzyme, the activity of the enzyme was detected by color development ofp-nitroaniline (Non-patent document 1), but generation of γ-Glu-Val-Glywas not confirmed.

GGT of Escherichia coli consists of two subunits, a large subunit and asmall subunit, and the ggt gene coding for GGT comprises ORFs (openreading frames) coding for a leader peptide, the large subunit, and thesmall subunit (Patent document 9). With advance of the transcription andtranslation of the ggt gene, transfer of the translation product to theperiplasm, cleavage of the leader peptide and processing for generatingthe large subunit and the small subunit occur to generate the matureGGT.

As for researches concerning the structural analysis of GGT, it wasreported that the D433N mutation (Non-patent document 2) or the Y444mutation and G484 mutation (Non-patent document 3) of Escherichia coliGGT impart a novel acylase activity, specifically an ability tohydrolyze glutaryl-7-aminocephalosporanic acid, to GGT, or improve suchan ability. Non-patent document 3 (Yamada et al.) specifically describesN411G, N411H, N411Y, Q430I, Q430V, D433A, D433G, D433L, D433N, Y444A,Y444F, Y444G, Y444H, Y444I, Y444L, Y444V, G484A, P460V, L461F, and S462Tmutations.

Further, as information concerning the structure around the activecenter of GGT, it was reported that Y444 in GGT of Escherichia coli(Non-patent document 3) or Y433 in GGT of Helicobacter Pylori(Non-patent document 4) locates in the lid-loop that covers thesubstrate-binding site.

Furthermore, it was also reported that Arg114 and Arg337 are importantfor the function of GGT of Escherichia coli (Non-patent document 5), andthis reference describes R114K, R114L, R114D, R337K, R337L, and R337Dmutations.

Further, it is also reported that most of mutants of Escherichia coliGGT having mutations at Thr407, Asp433, and Met464 in the small subunitlost the activity (Non-patent document 6). This reference describesN411G, N411H, N411Y, Q430I, Q430V, D433A, D433G, D433L, D433N, Y444A,Y444F, Y444G, Y444H, Y444I, Y444L, Y444V, G484A, P460V, L461F, and S462Tmutations.

In addition, influences of T391, T392, H393, Q390 and V396 mutations(Non-patent document 7), and R513 and R571 mutations (Non-patentdocument 8) on processing into the subunits of Escherichia coli GGT orthe GGT activity were reported. The former reference describes T391A,T391S, T392A, H393G, Q390A, and V396T mutations, and the latterreference describes R513A, R513G, R571A, and R571G mutations.

However, any mutation or combination of mutations preferred for theγ-glutamylation of Val-Gly is not known.

For the decomposition of Val-Gly, it was reported that PepA hydrolyzesVal-Gly (Non-patent document 9), and PepA- and PepN-deficientEscherichia coli does not decompose Val-Gly (Non-patent document 10),but activity of pepD for decomposing Val-Gly is not known.

PRIOR ART REFERENCES Patent Documents

-   Patent document 1: WO2007/055388-   Patent document 2: WO2007/055393-   Patent document 3: WO2008/139945-   Patent document 4: WO2008/139946-   Patent document 5: WO2008/139947-   Patent document 6: WO2009/107660-   Patent document 7: WO2009/119554-   Patent document 8: WO2004/011653-   Patent document 9: Japanese Patent Laid-open (Kokai) No. 02-231085-   Patent document 10: Japanese Patent Laid-open No. 08-119916

Non-Patent Documents

-   Non-patent document 1: Suzuki, H. et al. (2008) Improvement of the    flavor of amino acids and peptides using bacterial    γ-glutamyltranspeptidase, In Recent Highlights in Flavor Chemistry &    Biology, Ed. by Hofmann, T. et al., pp. 227-232, Deutsche    Forschungsanstalt fur Lebensmittelchemie-   Non-patent document 2: Suzuki, H. et al., Applied and Environmental    Microbiology, 70 (10), 6324-6328, 2004-   Non-patent document 3: Yamada, C. et al., Appl. Environ. Microbiol.,    74(11):3400-3409, 2008-   Non-patent document 4: Morrow, A. L. et al., Biochemistry., 20;    46(46):13407-13414, 2007-   Non-patent document 5: Ong, P. L. et al., Biochem. Biophys. Res.    Commun., 366(2):294-300, 2008-   Non-patent document 6: Lo, H. F. et al, Indian J. Biochem. Biophys.,    44(4):197-203, 2007-   Non-patent document 7: Hashimoto, W. et al., J. Biochem.,    118(1):75-80, 1995-   Non-patent document 8: Hashimoto, W. et al., Biochem. Biophys. Res.    Commun., 189(1):173-178, 1992-   Non-patent document 9: Gu, Y. Q. et al., Eur. J. Biochem.,    267:1178-1187, 2000-   Non-patent document 10: Miller, C. G. et al., Journal of    Baccteriol., 135(2):603-611, 1978

DISCLOSURE OF THE INVENTION Object to be Achieved by the Invention

An object of the present invention is to provide a mutant of GGTsuitable for the γ-glutamylation of Val-Gly, and a method for producingγ-Glu-Val-Gly or a salt thereof using such a mutant GGT.

Means for Achieving the Object

The inventor of the present invention conducted various researches inorder to achieve the aforementioned object, as a result, they foundmutations of GGT suitable for the γ-glutamylation of Val-Gly, andaccomplished the present invention.

The present invention thus relates to the followings.

(1) A method for producing γ-Glu-Val-Gly comprising the step of reactingVal-Gly with a γ-glutamyl group donor in the presence of aγ-glutamyltransferase, a microorganism containing the enzyme, or aprocessed product thereof to generate γ-Glu-Val-Gly, wherein:

the γ-glutamyltransferase consists of a large subunit and a smallsubunit, and the small subunit has the amino acid sequence of thepositions 391 to 580 of SEQ ID NO: 2 or the amino acid sequence having ahomology of 90% or more to the foregoing amino acid sequence, and has amutation for one or more residues corresponding to one or more residuesselected from the following residues in the amino acid sequence of SEQID NO: 2:

N411, T413, Q430, P441, V443, Y444, L446, A453, D472, G484, S498, Q542,D561, S572.

(2) The method as described above, wherein the mutation corresponds to amutation selected from the following mutations:

N411(Q) T413(H, N, A) Q430(M, N) P441A V443(E, L, G, N, Q, A) Y444(D, E,N, A) L446A A453S D472 (I) G484 (S, A, E) S498C Q542H D561N S572K.

(3) The method as described above, wherein the mutation is a mutationcorresponding to any one of the following mutations:

N411Q, Q430M, Y444A, Y444D, Y444E, G484S, (T413A+Y444E), (T413H+Y444E),(T413N+Y444E), (Q430N+Y444E), (Q430N+Y444D), (Q430N+Y444N),(P441A+Y444E), (V443A+Y444E), (V443E+Y444E), (V443G+Y444E),(V443L+Y444E), (V443N+Y444E), (V443Q+Y444E), (Y444E+L446A),(Y444E+A453S), (Y444E+D472I), (Y444E+G484A), (Y444E+G484S),(Y444E+S498C), (Y444E+Q542H), (Y444E+D561N), (T413N+Y444E+V443A),(T413N+Y444E+A453S), (T413N+Y444E+S498C), (T413N+Y444E+Q542H),(G484S+Y444E+V443A), (G484S+Y444E+Q542H), (Q430N+Y444E+T413N),(T413H+Y444E+G484S), (T413N+Y444E+G484S), (T413N+Y444E+G484S+V443A),(T413N+Y444E+G484S+A453S), (T413N+Y444E+G484S+Q542H),(T413N+Y444E+G484S+S572K) (T413N+Y444E+G484S+Q430N),(T413N+Y444E+G484E+S498C).

(4) The method as described above, wherein the large subunit has theamino acid sequence of the positions 26 to 390 of SEQ ID NO: 2 or theamino acid sequence having a homology of 90% or more to the foregoingamino acid sequence.

(5) The method as described above, wherein the large subunit has amutation corresponding to any one of the following mutations in theamino acid sequence of SEQ ID NO: 2:

P27H, E38K, L127V, F174A, F1741, F174L, F174M, F174V, F174W, F174Y,T246R, T276N, V301L.

(6) The method as described above, wherein the small subunit has theamino acid sequence of SEQ ID NO: 13 except for the aforementionedmutation.

(7) The method as described above, wherein the small subunit has theamino acid sequence of:

the positions 391 to 580 of SEQ ID NO: 2, the positions 391 to 580 ofSEQ ID NO: 3, the positions 392 to 581 of SEQ ID NO: 4, the positions388 to 577 of SEQ ID NO: 5, the positions 391 to 580 of SEQ ID NO: 6,the positions 391 to 580 of SEQ ID NO: 7, the positions 391 to 580 ofSEQ ID NO: 8, the positions 400 to 589 of SEQ ID NO: 9, the positions391 to 580 of SEQ ID NO: 10, the positions 392 to 581 of SEQ ID NO: 11,or the positions 392 to 581 of SEQ ID NO: 12, or

any one of these amino acid sequences including substitutions,deletions, insertions, additions, or inversions of one or several aminoacid residues, except for the aforementioned mutation.

(8) The method as described above, wherein the large subunit has theamino acid sequence of:

the positions 26 to 390 of SEQ ID NO: 2, the positions 26 to 390 of SEQID NO: 3, the positions 26 to 391 of SEQ ID NO: 4, the positions 26 to387 of SEQ ID NO: 5, the positions 25 to 390 of SEQ ID NO: 6, thepositions 25 to 390 of SEQ ID NO: 7, the positions 25 to 390 of SEQ IDNO: 8, the positions 33 to 399 of SEQ ID NO: 9, the positions 25 to 390of SEQ ID NO: 10, the positions 25 to 391 of SEQ ID NO: 11, or thepositions 25 to 391 of SEQ ID NO: 12, or

any one of these amino acid sequences including substitutions,deletions, insertions, additions, or inversions of one or several aminoacid residues, except for the aforementioned mutation.

(9) The method as described above, wherein the γ-glutamyl group donor isL-glutamine.

(10) The method as described above, wherein the γ-glutamyltransferase,the microorganism containing the enzyme, or the processed productthereof is a microorganism containing the enzyme, or a processed productthereof, and the microorganism is a bacterium belonging to the familyEnterobacteriaceae.

(11) The method as described above, wherein the microorganism is anEscherichia bacterium.

(12) The method as described above, wherein the microorganism isEscherichia coli.

(13) The method as described above, wherein the microorganism isdeficient in peptidase D.

(14) The method as described above, wherein the reaction is performed inthe presence of a metal chelating agent.

(15) A mutant γ-glutamyltransferase consisting of the following largesubunit and small subunit:

(A) a large subunit which has the amino acid sequence of the positions26 to 390 of SEQ ID NO: 2 or the amino acid sequence includingsubstitutions, deletions, insertions, additions, or inversions of one orseveral amino acid residues, and able to form a complex having theγ-glutamyltransferase activity with any one of the following smallsubunit;

(B) a small subunit which has the amino acid sequence of the positions391 to 580 of SEQ ID NO: 2 or the amino acid sequence includingsubstitutions, deletions, insertions, additions, or inversions of one orseveral amino acid residues, has any one of the following mutations, andable to form a complex having the γ-glutamyltransferase activity withthe above large subunit:

Y444D, Y444E, (T413A+Y444E), (T413H+Y444E), (T413N+Y444E),(Q430N+Y444E), (Q430N+Y444D), (Q430N+Y444N), (P441A+Y444E),(V443A+Y444E), (V443E+Y444E), (V443G+Y444E), (V443L+Y444E),(V443N+Y444E), (V443Q+Y444E), (Y444E+L446A), (Y444E+A453S),(Y444E+D472I), (Y444E+G484A), (Y444E+G484S), (Y444E+S498C),(Y444E+Q542H), (Y444E+D561N), (T413N+V443A+Y444E), (T413N+Y444E+A453S),(T413N+Y444E+S498C), (T413N+Y444E+Q542H), (G484S+Y444E+V443A),(G484S+Y444E+Q542H), (Q430N+Y444E+T413N), (T413H+Y444E+G484S),(T413N+Y444E+G484S), (T413N+Y444E+G484S+V443A),(T413N+Y444E+G484S+A453S), (T413N+Y444E+G484S+Q542H),(T413N+Y444E+G484S+S572K), (T413N+Y444E+G484S+Q430N),(T413N+Y444E+G484E+S498C).

(16) The mutant γ-glutamyltransferase as described above, wherein thelarge subunit has any one of the following mutations:

P27H, E38K, L127V, F174A, F1741, F174L, F174M, F174V, F174W, F174Y,T246R, T276N, V301L.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows alignment of the amino acid sequences of the GGT smallsubunits of various bacteria. The strains of the bacteria are asfollows. The numerals described before and after the amino acidsequences represent positions from the N-terminus of the small subunit.Relations of the amino acid sequences and the amino acid sequencesdescribed in Sequence Listing are also shown.

Consensus: consensus sequence (SEQ ID NO: 13)

E. coli: Escherichia coli (SEQ ID NO: 2, 391 to 580)

Sh. flexneri 5 str. 8401: Shigella flexneri 5 str. 8401 (SEQ ID NO: 3,391 to 580)

Sh. dysenteriae Sd197: Shigella dysenteriae Sd197 (SEQ ID NO: 4, 392 to581)

Sh. boydii Sb227: Shigella boydii strain Sb227 (SEQ ID NO: 5, 388 to577)

S. typhimurium ATCC700720: Salmonella enterica typhimurium strain ATCC700720 (also designated as Salmonella typhimurium LT2, SEQ ID NO: 6, 391to 580)

S. enterica SC-B67: Salmonella enterica enterica choleraesuis strainSC-B67 (SEQ ID NO: 7, 391 to 580)

S. typhi Ty2: Salmonella enterica typhi strain Ty2 (SEQ ID NO: 8, 391 to580)

K. pneumoniae ATCC202080: Klebsiella pneumoniae strain ATCC 202080 (SEQID NO: 9, 400 to 589)

S. enterica ATCC 9150: Salmonella enterica subsp. enterica serovarParatyphi A str. ATCC 9150 (SEQ ID NO: 10, 391 to 580)

K. pneumoniae KPN308894: Klebsiella pneumoniae clone KPN 308894 (SEQ IDNO: 11, 392 to 581)

En. cloaceae EBC103795: Enterobacter cloaceae clone

EBC103795 (SEQ ID NO: 12, 392 to 581)

FIG. 2 shows continuation of FIG. 1.

EMBODIMENTS FOR CARRYING OUT THE INVENTION

Hereafter, the present invention will be explained in detail.

The method for producing γ-Glu-Val-Gly or a salt thereof of the presentinvention comprises the step of reacting Val-Gly or a salt thereof witha γ-glutamyl group donor in the presence of GGT, a microorganismcontaining the enzyme, or a processed product thereof to generateγ-Glu-Val-Gly or a salt thereof. The method of the present invention ischaracterized in that GGT consists of a large subunit and a smallsubunit, and at least the small subunit has a specific mutation.Hereafter, GGT having such a specific mutation and the method forproducing γ-Glu-Val-Gly or a salt thereof using it will be explained.

In this specification, amino acids are L-amino acids, unless especiallymentioned.

<1> Mutant GGT

GGT having the aforementioned specific mutation (also referred to as“mutant GGT”) can be obtained by modifying a ggt gene coding for GGT nothaving the specific mutation, so that the encoded GGT has the specificmutation, and expressing the obtained modified ggt gene. GGT not havingthe aforementioned specific mutation may be referred to as a wild-typeGGT, and a ggt gene coding for the wild-type GGT may be referred to as awild-type ggt gene. The wild-type GGT may have other mutations, so longas it does not have the specific mutation. The specific mutation will beexplained later.

Examples of the wild-type GGT include GGT encoded by the ggt gene ofEscherichia coli and homologues thereof, for example, GGT of Escherichiacoli, and GGTs of other microorganisms, especially those of which smallsubunit has a similar structure.

The nucleotide sequence of the ggt gene of the Escherichia coli K-12strain is described in Japanese Patent Laid-open No. 02-231085. Further,the nucleotide sequence of the ggt gene of the Escherichia coli K-12W3110 strain is registered in the database as 4053592.4055334 of GenBankaccession AP009048. The nucleotide sequence of this ggt gene is shown inSEQ ID NO: 1. Further, the amino acid sequence encoded by thisnucleotide sequence is shown in SEQ ID NO: 2. In SEQ ID NO: 2, thepositions 1 to 25 correspond to the leader peptide, the positions 26 to390 correspond to the large subunit, and the positions 391 to 580correspond to the small subunit.

As GGT homologues homologous to GGT of Escherichia coli, thosecontaining a small subunit having an amino acid sequence showing ahomology of 90% or more to the site corresponding to the small subunitin the amino acid sequence shown in SEQ ID NO: 2 (positions 391 to 580)are preferred. As the GGT homologues, those containing a large subunithaving an amino acid sequence showing a homology of 90% or more to thesite corresponding to the large subunit in the amino acid sequence shownin SEQ ID NO: 2 (positions 26 to 390) are preferred. Specific examplesinclude GGTs of bacteria belonging to the family Enterobacteriaceae.Although the bacteria belonging to the family Enterobacteriaceae are notparticularly limited, they include bacteria belonging to the genera ofEscherichia, Enterobacter, Erwinia, Klebsiella, Pantoea, Photorhabdus,Providencia, Salmonella, Serratia, Shigella, Morganella, Yersinia, andso forth. In particular, bacteria classified into the familyEnterobacteriaceae according to the taxonomy used in the NCBI (NationalCenter for Biotechnology Information) database(http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?id=91347) arepreferred. Specific examples include, for example, Shigella flexneri,Shigella dysenteriae, Shigella boydii, Salmonella typhimurium,Klebsiella pneumoniae, Salmonella enterica, Enterobacter cloacae, and soforth. The amino acid sequences of GGTs of Shigella flexneri 5 str. 8401(GenBank accession ABF05491), Shigella dysenteriae Sd197 (GenBankaccession ABB63568), Shigella boydii Sb227 (GenBank accession ABB67930),Salmonella enterica typhimurium strain ATCC 700720 (also designated asSalmonella typhimurium LT2, GenBank accession AAL22411), Salmonellaenterica enterica choleraesuis strain SC-B67 (GenBank accessionAAX67386), Salmonella enterica typhi strain Ty2 (GenBank accessionAAO71440), Klebsiella pneumoniae ATCC 202080 (U.S. Pat. No. 6,610,836,SEQ ID NO: 10810), Salmonella enterica subsp. enterica serovar ParatyphiA str. ATCC 9150 (GenBank accession AAV79214), Klebsiella pneumoniaeclone KPN308894 (WO02/077183, SEQ ID NO: 60310), and Enterobactercloacae clone EBC103795 (WO02/077183, SEQ ID NO: 56162) are shown in SEQID NOS: 3 to 12.

The positions of the leader peptide, the large subunit, and the smallsubunit in each GGT are shown in Table 1. Further, alignment of theamino acid sequences of the small subunits of those GGTs is shown inFIGS. 1 and 2. The consensus sequence of the small subunit amino acidsequences is shown in FIGS. 1 and 2 and SEQ ID NO: 13. Further,identities of the small subunits of those bacteria and the small subunitof Escherichia coli are shown in Table 1. The amino acid codes used inFIGS. 1 and 2 are as follows. Specific examples of X include the aminoacid residues locating at positions corresponding to the positions of Xin the GGT small subunits of the bacteria described in the alignmentshown in FIGS. 1 and 2. For example, X at the position 4 of SEQ ID NO:13 is Tyr or Phe.

A: Ala, C: Cys, D: Asp, E: Glu, F: Phe, G: Gly, H: His, I: Ile, K: Lys,L: Leu, M: Met, N: Asn, P: Pro, Q: Gln, R: Arg, S: Ser, T: Thr, V: Val,W: Trp, X: Xaa, Y: Tyr

TABLE 1 Identity SEQ of small ID Leader Large Small subunit Bacterium NOpeptide subunit subunit (%) Escherichia coli 2 1-25 26-390 391-580Shigella flexneri 5 str. 3 1-25 26-390 391-580 99 8401 Shigelladysenteriae Sd197 4 1-25 26-391 392-581 99 Shigella boydii strain 5 1-2526-387 388-577 99 Sb227 Salmonella enterica 6 1-24 25-390 391-580 95typhimurium strain ATCC 700720 Salmonella enterica 7 1-24 25-390 391-58095 enterica choleraesuis strain SC-B67 Salmonella enterica typhi 8 1-2425-390 391-580 95 strain Ty2 Klebsiella pneumoniae 9 1-32 33-399 400-58995 strain ATCC 202080 Salmonella enterica subsp. 10 1-24 25-390 391-58094 enterica serovar Paratyphi A str. ATCC 9150 Klebsiella pneumoniae 111-24 25-391 392-581 95 clone KPN308894 Enterobacter cloaceae 12 1-2425-391 392-581 93 clone EBC103795

Preferred examples of the wild-type GGT are those having the amino acidsequence of SEQ ID NO: 13 in the small subunit.

The mutant GGT of the present invention has one or more mutations at oneor more residues selected from the following residues in the smallsubunit:

N411, T413, Q430, P441, V443, Y444, L446, A453, D472, G484, S498, Q542,D561, 5572.

In the above indications, the letters on the left side of the numeralsrepresent type of amino acid residue, and the numerals representposition in GGT. The position of amino acid residue is represented asthe position on the amino acid sequence of the GGT precursor (proteinconsisting of the leader peptide, the large subunit, and the smallsubunit connected in this order) encoded by ORFs of the ggt gene. Forexample, the amino acid residue of the N-terminus of the GGT smallsubunit of Escherichia coli corresponds to the position 26 of SEQ ID NO:2. The term amino acid sequence of GGT may henceforth mean the aminoacid sequence of the GGT precursor, unless especially indicated.

The amino acid residue existing at the position of the mutation afterthe substitution may be any amino acid residue so long as it is an aminoacid residue other than the original amino acid residue, and specificexamples of the mutation include those selected from the followingmutations:

N411Q T413(H, N, A) Q430(M, N) P441A V443(E, L, G, N, Q, A) Y444(D, E,N, A) L446A A453S D472I G484(S, A, E) S498C Q542H D561N S572K.

The meanings of the letters representing the type of amino acid in theindications of the mutations are the same as those described above. Thenumerals represent the positions of the mutation. The letters on theleft side of the numerals represent the amino acid residues existing inthe wild-type, and the letters on the right side of the numeralsrepresent the amino acid residue existing after the mutation. Forexample, “N411Q” means a substitution of Gln residue for the Asn residueat the position 411. Further, the letters in the parentheses on theright side of the numerals collectively represent amino acid residuesexisting after the mutation. For example, T413(H, N, A) meanssubstitution of His, Asn or Ala residue for the Thr residue at theposition 413.

More specific examples of the mutant GGT of the present inventioninclude those having any one of the following mutations in the smallsubunit. The collective indications of two or more mutations using thesymbol “+” means a double mutation or a more multiple mutation. Forexample, (N411Q+Q430N) means that the mutant GGT simultaneously has theN411Q mutation and the Q430N mutation.

N411Q, T413H, T413N, T413A, Q430M, Q430N, P441A, V443E, V443L, V443G,V443N, V443Q, V443A, Y444D, Y444E, Y444N, Y444A, L446A, A453S, D472I,G484S, G484A, G484E, S498C, Q542H, D561N, S572K,

(N411Q+Q430N), (N411Q+S572K) (T413N+G484E) (T413N+G484S) (T413A+Y444E)(T413H+Y444E) (T413N+Y444E) (Q430N+Y444E) (Q430N+Y444D) (Q430N+Y444N)(P441A+Y444E) (V443A+Y444E) (V443E+Y444E) (V443G+Y444E) (V443L+Y444E)(V443N+Y444E) (V443Q+Y444E) (V443A+Y444E) (Y444E+L446A) (Y444E+A453S)(Y444E+D472I) (Y444E+G484A) (Y444E+G484S) (Y444E+Q542H) (Y444E+D561N)(T413N+Y444E+V443A) (T413N+Y444E+A453S) (T413N+Y444E+S498C)(T413N+Y444E+Q542H) (G484S+Y444E+T276N) (G484S+Y444E+V443A)(G484S+Y444E+Q542H) (Q430N+Y444E+T413N) (T413H+Y444E+G484S)(T413N+Y444E+G484S) (T413N+Y444E+G484S+V443A) (T413N+Y444E+G484S+A453S)(T413N+Y444E+G484S+Q542H) (T413N+Y444E+G484S+S572K)(T413N+Y444E+G484S+Q430N) (T413N+Y444E+G484E+S498C)

As for the aforementioned mutations, the positions 413, 472, and 572 arenovel mutation sites, and N411Q, Q430M, Q430N, Q430P, Q430S, Q430Y,Y444D, Y444E, D472S, and G484S are novel mutations.

Among the aforementioned mutations, preferred are those with whichgeneration of γ-Glu-Val-Gly was confirmed in Example 5. Further, thoseespecially preferred for the method of the present invention are thosewith which γ-Glu-Val-Gly generation amount of 40 mM or more concerning asingle mutation, or 60 mM or more concerning a complex mutation wasobtained in Example 5. For example, the enzyme having N411Q, Q430M,Y444A, Y444D, Y444E, G484S, a double mutation, triple mutation, orquadruple mutation of the foregoing mutations is preferred.

Further, from another aspect, the Y444E mutation or a complex mutationof the Y444E mutation and one or more other mutations among theaforementioned mutations is also preferred.

Furthermore, as the mutant γ-glutamyltransferase of the presentinvention, especially preferred are mutant enzymes showing a higheractivity compared with the known mutant γ-glutamyltransferase having theY444A mutation (Glu-Val-Gly production amount observed in Example 5 is52.5 mM), such as the enzymes having Y444D, Y444E, a double mutation,triple mutation, or quadruple mutation of the aforementioned mutations.

As for GGTs of microorganisms other than Escherichia coli, the positionsof the mutations thereof corresponding to the mutations on the aminoacid sequence of GGT of Escherichia coli are represented by thecorresponding positions in GGT of Escherichia coli determined inalignment of the amino acid sequences of the GGTs of the othermicroorganisms and the amino acid sequence of GGT of Escherichia coli.For example, an amino acid residue of a position 100 in an amino acidsequence of GGT of a certain microorganism corresponds to the position101 of the amino acid sequence of GGT of Escherichia coli in thealignment, that amino acid residue of the position 100 is regarded asthe amino acid residue of the position 101. In the present invention, aresidue corresponding to a specific residue in SEQ ID NO: 2 means aresidue corresponding to the specific amino acid residue in the aminoacid sequence of SEQ ID NO: 2 in the alignment of the amino acidsequence of SEQ ID NO: 2 and an objective sequence, as described above.Similarly, a mutation corresponding to a specific mutation in SEQ ID NO:2 is a mutation at a residue corresponding to a residue of the specificmutation in the amino acid sequence of SEQ ID NO: 2 in the alignment ofthe amino acid sequence of SEQ ID NO: 2 and an objective sequence, asdescribed above.

As a means for performing the alignment, known gene analysis softwarecan be used. Specific examples of such software include DNASIS producedby Hitachi Solutions, GENETYX produced by Genetyx, and so forth(Elizabeth C. Tyler et al., Computers and Biomedical Research, 24(1),72-96, 1991; Barton G J et al., Journal of Molecular Biology, 198(2),327-37, 1987).

The positions of the mutations do not necessarily represent the absolutepositions from the N-terminus in the amino acid sequences of mutantGGTs, and represent relative positions with respect to the amino acidsequence shown in SEQ ID NO: 2. For example, if one amino acid residueof GGT having the amino acid sequence shown in SEQ ID NO: 2 is deletedat a position on the N-terminus side with respect to a position n, thisposition n becomes an (n−1) position from the N-terminus. However, evenin such a case, the amino acid residue of this position is regarded asthe amino acid residue of the position n. An absolute position of anamino acid substitution can be determined on the basis of alignment ofan amino acid sequence of an objective GGT and the amino acid sequenceof SEQ ID NO: 2. The method for performing the alignment for this caseis the same as the method described above.

Although the mutant GGT of the present invention may not contain amutation in the large subunit, it may have any of the mutations shownbelow:

P27H, E38K, L127V, F174A, F1741, F174L, F174M, F174V, F174W, F174Y,T246R, T276N, V301L.

Examples of the combination of the mutations of the small subunit andthe large subunit include the following combinations:

(E38K⁺ Y444E) (F174A+Y444E) (F1741+Y444E) (F174L+Y444E) (F174M+Y444E)(F174V+Y444E) (F174W+Y444E) (F174Y+Y444E) (T246R+Y444E) (V301L+Y444E)(T413N+Y444E+P27H) (T413N+Y444E+E38K) (T413N+Y444E+L127V)(T413N+Y444E+T276N) (G484S+Y444E+T276N) (G484S+Y444E+P27H)

Furthermore, the mutant GGT of the present invention may be aconservative variant of the proteins having the aforementioned aminoacid sequences, i.e., a homologue, an artificially modified protein orthe like of the proteins concerning the amino acid sequence thereof,such as any of the amino acid sequences of SEQ ID NOS: 2 to 12, so longas the GGT activity is not degraded. That is, the mutant GGT of thepresent invention may have any of the aforementioned amino acidsequences including, in addition to the aforementioned specificmutations, substitutions, deletions, insertions, additions, orinversions of one or several amino acid residues. Although the numbermeant by the term “one or several” can differ depending on the positionsof amino acid residues in the three-dimensional structure or the typesof amino acid residues of the protein, specifically, it is preferably 1to 20, more preferably 1 to 10, still more preferably 1 to 5. Theconservative mutation is typically a conservative substitution. Theconservative substitution is a mutation wherein substitution takes placemutually among Phe, Trp, and Tyr, if the substitution site is anaromatic amino acid; among Leu, Ile and Val, if the substitution site isa hydrophobic amino acid; between Gln and Asn, if the substitution siteis a polar amino acid; among Lys, Arg and His, if the substitution siteis a basic amino acid; between Asp and Glu, if the substitution site isan acidic amino acid; and between Ser and Thr, if the substitution siteis an amino acid having a hydroxyl group. Substitutions consideredconservative substitutions include, specifically, substitution of Ser orThr for Ala, substitution of Gln, His or Lys for Arg, substitution ofGlu, Gln, Lys, His or Asp for Asn, substitution of Asn, Glu or Gln forAsp, substitution of Ser or Ala for Cys, substitution of Asn, Glu, Lys,His, Asp or Arg for Gln, substitution of Gly, Asn, Gln, Lys or Asp forGlu, substitution of Pro for Gly, substitution of Asn, Lys, Gln, Arg orTyr for His, substitution of Leu, Met, Val or Phe for Ile, substitutionof Ile, Met, Val or Phe for Leu, substitution of Asn, Glu, Gln, His orArg for Lys, substitution of Ile, Leu, Val or Phe for Met, substitutionof Trp, Tyr, Met, Ile or Leu for Phe, substitution of Thr or Ala forSer, substitution of Ser or Ala for Thr, substitution of Phe or Tyr forTrp, substitution of His, Phe or Trp for Tyr, and substitution of Met,Ile or Leu for Val. The aforementioned amino acid substitutions,deletions, insertions, additions, inversions or the like can be a resultof a naturally-occurring mutation due to an individual difference,difference of species, or the like of a microorganism from which thegene is derived (mutant or variant).

Further, GGT having such a conservative mutation as described above canbe a protein showing a homology of, for example, 80% or more, preferably90% or more, more preferably 95% or more, still more preferably 97% ormore, particularly preferably 99% or more, to any of the amino acidsequences of SEQ ID NOS: 2 to 12, and having the GGT activity.

In this specification, “homology” can mean “identity”.

Further, GGT may be a protein encoded by a DNA that is able to hybridizewith a prove having a nucleotide sequence complementary to thenucleotide sequence of SEQ ID NO: 1, or a probe that can be preparedfrom the complementary sequence under stringent conditions, and havingthe GGT activity. The “stringent conditions” refer to conditions underwhich a so-called specific hybrid is formed, and a non-specific hybridis not formed. Examples of the stringent conditions include those underwhich highly homologous DNAs hybridize to each other, for example, DNAsnot less than 80% homologous, preferably not less than 90% homologous,more preferably not less than 95% homologous, still more preferably notless than 97% homologous, particularly preferably not less than 99%homologous, hybridize to each other, and DNAs less homologous than theabove do not hybridize to each other, for example, conditions ofhybridization at 42° C. and washing with a buffer containing 1×SSC and0.1% SDS at 42° C., more preferably conditions of hybridization at 65°C. and washing with a buffer containing 0.1×SSC and 0.1% SDS at 65° C.The factors affecting the stringency for hybridization include variousfactors other than the aforementioned temperature conditions, and thoseskilled in the art can realize a stringency corresponding to thestringency exemplified above by using an appropriate combination of thevarious factors.

The probe used for the hybridization may be a part of a complementarysequence of the ggt gene. Such a probe can be produced by PCR usingoligonucleotides synthesized on the basis of a known gene sequence asprimers and a DNA fragment containing the nucleotide sequence as atemplate. For example, when a DNA fragment having a length of about 300bp is used as the probe, the washing conditions of the hybridization canbe, for example, 50° C., 2×SSC and 0.1% SDS.

The mutant GGT of the present invention can be produced by inserting amutant ggt gene coding for it into an appropriate vector, andintroducing the obtained recombinant vector into an appropriate host toallow expression thereof. Further, a microorganism containing the mutantGGT used for the method for producing γ-Glu-Val-Gly described later canbe obtained by introducing the recombinant vector into an appropriatehost microorganism.

The ggt gene having the specific mutation can be obtained by, forexample, modifying a nucleotide sequence of a wild-type ggt gene, forexample, a ggt gene coding for any of the amino acid sequences of SEQ IDNOS: 2 to 12, by the site-directed mutagenesis method, so that the aminoacid residue of the specific position of the encoded GGT is replacedwith another amino acid residue.

Examples of the site-directed mutagenesis method for introducing anobjective mutation at an intended site of DNA include, for example, amethod using PCR (Higuchi, R., 61, in PCR technology, Erlich, H. A.Eds., Stockton Press, 1989; Carter P., Meth. In Enzymol., 154, 382,1987), as well as a method of using a phage (Kramer, W. and Frits, H.J., Methods in Enzymology, 154, 350, 1987; Kunkel, T. A. et al., Methodsin Enzymology, 154, 367, 1987), and so forth.

The vector into which a mutant ggt gene is incorporated is notparticularly limited so long as a vector that can replicate in the hostis chosen. When Escherichia coli is used as the host, examples of such avector include plasmids that can autonomously replicate in thisbacterium. For example, pUC19, pET, pGEMEX, pGEM-T and so forth can beused. Preferred examples of the host include Escherichia coli strains.However, other than these, any of microorganisms in which a replicationorigin and a mutant ggt gene of a constructed recombinant DNA canfunction, a recombinant DNA can be expressed, and the mutant ggt genecan be expressed can be used as the host. As the host, for example,Gram-negative bacteria including Escherichia bacteria such asEscherichia coli, Enterobacter bacteria, Pantoea bacteria, and so forth,and Gram-positive bacteria including Bacillus bacteria, Corynebacteriumbacteria, and so forth can be used. For example, Bacillus subtilis isknown to secrete produced GGT out of cells (Xu et al., Journal ofBacteriology, Vol. 178, No. 14, 1996), and a mutant GGT may be secretedout of cells. In addition, an objective mutant ggt gene may be expressedby using cells of yeast, mold, or the like.

Examples of transformation methods include treating recipient cells withcalcium chloride to increase permeability for DNA, which has beenreported for Escherichia coli K-12 (Mandel, M. and Higa, A., J. Mol.Biol., 53:159-162, 1970), preparing competent cells from cells which areat the growth phase, followed by transformation with DNA, which has beenreported for Bacillus subtilis (Duncan, C. H., Wilson, G. A. and Young,F. E., Gene, 1:153-167, 1977), and so forth. Alternatively, a method ofmaking DNA-recipient cells into protoplasts or spheroplasts, which caneasily take up recombinant DNA, followed by introducing a recombinantDNA into the cells, which is known to be applicable to Bacillussubtilis, actinomycetes and yeasts (Chang, S, and Choen, S. N., Mol.Gen. Genet., 168:111-115, 1979; Bibb, M. J., Ward, J. M. and Hopwood, O.A., Nature, 274:398-400, 1978; Hinnen, A., Hicks, J. B. and Fink, G. R.,Proc. Natl. Sci., USA, 75:1929-1933, 1978) can also be employed. Inaddition, transformation of microorganisms can also be performed by theelectroporation method (Japanese Patent Laid-open No. 2-207791).

The promoter for expressing the mutant ggt gene may be a promoterinherent to the ggt gene, or a promoter of another gene. Examples ofpromoters of other genes include rpoH promoter, lac promoter, trppromoter, trc promoter, tac promoter, PR promoter and PL promoter oflambda phage, tet promoter, and so forth.

Further, as a vector into which the ggt gene is inserted, an expressionvector containing a promoter suitable for gene expression may also beused.

A transformant introduced with the recombinant DNA containing the mutantggt gene obtained as described above can be cultured in an appropriatemedium containing a carbon source, a nitrogen source, inorganic ions,and organic nutrients if needed to allow expression of the mutant GGT.

When a microorganism that expresses a mutant ggt gene is used forproducing γ-Glu-Val-Gly, the microorganism is preferably deficient inthe peptidase D (PepD). The term “deficient in PepD” means that themicroorganism is completely deficient in PepD, or the microorganism hasa reduced amount or activity of PepD compared with a wild-type strain.The inventors of the present invention found that PepD is deeplyinvolved in the decomposition of Val-Gly in Escherichia coli. By makinga microorganism that expresses a mutant GGT deficient in PepD, thegeneration amount of γ-Glu-Val-Gly, which is generated from Val-Gly as asubstrate, can be increased.

The microorganism can be made deficient in PepD by, for example,reducing expression of the pepD gene coding for PepD. The expression ofthe pepD gene can be reduced by modifying the pepD gene on a chromosomeso that a wild-type RNA or wild-type protein is not expressed, forexample, by disrupting the pepD gene. As methods for such genedisruption, there are the method utilizing a linear DNA such as themethod called “Red-driven integration” (Datsenko, K. A. and Wanner, B.L., Proc. Natl. Acad. Sci. USA, 97:6640-6645, 2000), and the methodbased on the combination of the Red-driven integration method and the λphage excision system (Cho, E. H., Gumport, R. I., Gardner, J. F., J.Bacteriol., 184:5200-5203, 2002) (refer to WO2005/010175), the methodsutilizing a plasmid having a temperature sensitive replication origin, aplasmid capable of conjugative transfer, a suicide vector not having areplication origin in a host, and so forth (U.S. Pat. No. 6,303,383, orJapanese Patent Laid-open No. 05-007491).

The nucleotide sequence of the pepD gene of Escherichia coli and theamino acid sequence encoded by this gene are shown in SEQ ID NOS: 14 and15, respectively. Further, the nucleotide sequence of pepA, pepB, pepE,and pepN genes, which are other peptidase genes of Escherichia coli, areshown in SEQ ID NOS: 16, 18, 20, and 22, respectively. Further, theamino acid sequences encoded by these genes are shown in SEQ ID NOS: 17,19, 21, and 23, respectively. So long as the microorganism thatexpresses a mutant ggt gene is deficient in PepD, it may be deficient inanother arbitrary peptidase.

<2> Method for Producing γ-Glu-Val-Gly or Salt Thereof.

By reacting Val-Gly or a salt thereof with a γ-glutamyl group donor inthe presence of the mutant GGT obtained as described above, amicroorganism containing the mutant GGT, or a processed product thereofto generate γ-Glu-Val-Gly or a salt thereof, γ-Glu-Val-Gly or a saltthereof can be produced.

A microorganism containing the mutant GGT can be produced by culturing amicroorganism into which the mutant ggt gene has been introduced in anexpressible form under conditions enabling expression of the gene toallow growth of cells. The medium used for the culture is notparticularly limited so long as the objective microorganism can grow init, and there can be used a conventional medium containing a carbonsource, a nitrogen source, a sulfur source, inorganic ions, and otherorganic components as required.

As the carbon source, saccharides such as glucose, fructose, sucrose,glycerol, ethanol, molasses and starch hydrolysate, and organic acidssuch as fumaric acid, citric acid and succinic acid can be used.

As the nitrogen source, inorganic ammonium salts such as ammoniumsulfate, ammonium chloride and ammonium phosphate, organic nitrogen suchas soybean hydrolysate, ammonia gas, aqueous ammonia and so forth can beused.

Examples of the sulfur source include inorganic sulfur compounds, suchas sulfates, sulfites, sulfides, hyposulfites and thiosulfates.

As organic trace amount nutrients, it is desirable to add requiredsubstances such as vitamin B₁, yeast extract and so forth in appropriateamounts. Other than these, potassium phosphate, magnesium sulfate, ironions, manganese ions and so forth are added in small amounts.

The culture conditions can be appropriately chosen according to themicroorganism to be used. The culture is preferably performed at aculture temperature of, for example, 20 to 45° C., preferably 24 to 45°C. The culture is preferably performed as aeration culture at an oxygenconcentration of 5 to 50%, desirably about 10%, with respect to thesaturated concentration. Further, pH during the culture is preferably 5to 9. For adjusting pH, inorganic or organic acidic or alkalinesubstances, such as calcium carbonate, ammonia gas, and aqueous ammonia,can be used.

By culturing the microorganism preferably for about 10 to 120 hoursunder such conditions as described above, the mutant GGT is accumulatedin the periplasm of cells.

In addition, by appropriately choosing the host to be used and designingthe ggt gene, it is also possible to accumulate GGT in cells or produceGGT with allowing secretion thereof out of cells.

The mutant GGT may be used in a state of being contained in cells, ormay be used as a crude enzyme fraction extracted from the cells or apurified enzyme. The mutant GGT can be extracted by the same method asthose for conventional extraction of a periplasmic enzyme, for example,osmotic shock method, freezing and thawing method, and so forth.Further, the mutant GGT can be purified by an appropriate combination ofmethods usually used for purification of enzyme, such as ammoniumsulfate fractionation, ion exchange chromatography, hydrophobicchromatography, affinity chromatography, gel filtration chromatography,and electrofocusing. When GGT is produced and secreted out of cells, themutant GGT collected from the medium can be used.

The processed product of the microorganism containing mutant GGT is notparticularly limited so long as it contains the mutant GGT in a statethat the mutant GGT can function, and examples include disrupted cells,cell extract, partially purified products thereof, purified enzyme, andso forth, as well as cells immobilized with acrylamide, carrageenan, orthe like, immobilized enzymes comprising the mutant GGT immobilized on asolid phase such as resin, and so forth.

In the presence of the mutant GGT, a microorganism containing the mutantGGT or a processed product thereof obtained as described above, Val-Glyand a γ-glutamyl group donor are reacted.

Val-Gly or a salt thereof can be produced by a chemical synthesis methodusing formyl-L-valine and glycine ethyl ester as the starting materials(Journal of the American Chemical Society, 80, 1154-1158, 1958).Alternatively, it is also possible to use a chemical synthesis methodusing N-carboxyanhydride of valine (valine-NCA) and glycine as thestarting materials (Canadian Journal of Chemistry, 51 (8), 1284-87,1973). Further, other methods known as peptide synthesis methods canalso be used (“Fundamentals and Experiments of Peptide Synthesis”,Maruzen Co., Ltd., 1985).

Further, when Val-Gly to be used is in the form of a salt, it may be anysalt so long as a chemically acceptable salt is used. Specific examplesof the “chemically acceptable salt” include, for acidic groups such ascarboxyl group, ammonium salt, salts with alkali metals such as sodiumand potassium, salts with alkaline earth metals such as calcium andmagnesium, aluminum salts, zinc salts, salts with organic amines such astriethylamine, ethanolamine, morpholine, pyrrolidine, piperidine,piperazine, and dicyclohexylamine, salts with basic amino acids such asarginine and lysine, and for basic groups, salts with inorganic acidssuch as hydrochloric acid, sulfuric acid, phosphoric acid, nitric acid,and hydrobromic acid, salts with organic carboxylic acids such as aceticacid, citric acid, benzoic acid, maleic acid, fumaric acid, tartaricacid, succinic acid, tannic acid, butyric acid, hibenzic acid, pamoicacid, enanthic acid, decanoic acid, teoclic acid, salicylic acid, lacticacid, oxalic acid, mandelic acid, and malic acid, and salts with organicsulfonic acid such as methanesulfonic acid, benzenesulfonic acid, andp-toluenesulfonic acid.

Furthermore, when γ-Glu-Val-Gl obtained by the method of the presentinvention is in the form of a salt, it may be a chemically andpharmaceutically acceptable edible salt, and examples include, foracidic groups such as carboxyl group, ammonium salts, salts with alkalimetals such as sodium and potassium, salts with alkaline earth metalssuch as calcium and magnesium, aluminum salts, zinc salts, salts withorganic amines such as triethylamine, ethanolamine, morpholine,pyrrolidine, piperidine, piperazine, and dicyclohexylamine, salts withbasic amino acids such as arginine and lysine, and for basic groups,salts with inorganic acids such as hydrochloric acid, sulfuric acid,phosphoric acid, nitric acid, and hydrobromic acid, salts with organiccarboxylic acids such as acetic acid, citric acid, benzoic acid, maleicacid, fumaric acid, tartaric acid, succinic acid, tannic acid, butyricacid, hibenzic acid, pamoic acid, enanthic acid, decanoic acid, teoclicacid, salicylic acid, lactic acid, oxalic acid, mandelic acid, and malicacid, and salts with organic sulfonic acid such as methanesulfonic acid,benzenesulfonic acid, and p-toluenesulfonic acid. When it is used forfoods, it is sufficient that it is an edible salt.

Peptide production using an enzymatic reaction can be performed by usingany of the following methods reported as methods for producing apeptide, namely, a condensation reaction using an N-protected andC-non-protected carboxyl component and an N-non-protected andC-protected amine component (reaction 1), a substitution reaction usingan N-protected and C-protected carboxyl component and an N-non-protectedand C-protected amine component (reaction 2), a substitution reactionusing an N-non-protected and C-protected carboxyl component and anN-non-protected and C-protected amine component (reaction 3), asubstitution reaction using an N-non-protected and C-protected carboxylcomponent and an N-non-protected and C-non-protected amine component(reaction 4), or a transfer reaction using an N-non-protected andC-protected carboxyl component and an N-non-protected andC-non-protected amine component (reaction 5), and purifying Val-Gly or asalt thereof from the reaction product.

Examples of the reaction 1 include, for example, the method forproducing Z-aspartylphenylalanine methyl ester from Z-aspartic acid andphenylalanine methyl ester (Japanese Patent Laid-open No. 53-92729),examples of the reaction 2 include, for example, the method forproducing acetylphenylalanylleucinamide from acetylphenylalanine ethylester and leucinamide (Biochemical J., 163, 531, 1977), examples of thereaction 3 include, for example, the method for producingarginylleucinamide from arginine ethyl ester and leucinamide(WO90/01555), examples of the reaction 4 include, for example, themethod for producing tyrosylalanine from tyrosine ethyl ester andalanine (EP 278787 A1, EP 359399 B1), and examples of the reaction 5include, for example, the method for producing alanylglutamine fromalanine methyl ester and glutamine (W2004/011653). It is possible toapply the above reactions to the production of Val-Gly or a saltthereof. In such a case, by reacting valine having an esterified oramidated carboxyl group and glycine having free amino group in thepresence of a peptide-producing enzyme, and purifying Val-Gly from thereaction product, Val-Gly can be produced.

The γ-glutamyl group donor can be chosen from γ-glutamyl compounds.Examples include, for example, glutamine, glutamic acid γ-alkyl esterssuch as glutamic acid γ-methyl ester, salts thereof, and so forth. Amongthese, glutamine and a salt thereof are preferred. This salt may also besuch a chemically acceptable salt as explained above, and the definitionthereof is the same as described above.

The reaction of Val-Gly or a salt thereof and the γ-glutamyl group donormay be performed by the batch method or the column method. When thebatch method is used, Val-Gly or a salt thereof, the γ-glutamyl groupdonor and the mutant GGT, a microorganism containing the mutant GGT, ora processed product thereof can be mixed in a reaction mixture containedin a reaction vessel. The reaction may be performed as a standingreaction, or with stirring. When the column method is used, a reactionmixture containing Val-Gly or a salt thereof and the γ-glutamyl groupdonor can be passed thorough a column filled with such immobilized cellsor immobilized enzyme as described above.

The reaction mixture preferably consists of water or a buffer containingVal-Gly or a salt thereof and the γ-glutamyl group donor, and preferablyhas pH of 6.0 to 10.0, more preferably 6.5 to 9.0.

The reaction time or the flow rate of the reaction mixture can beappropriately determined according to the concentrations of thesubstrates, amount of the mutant GGT with respect to the substrates, andso forth. Specifically, for example, the amount of the enzyme to beadded can be determined by measuring the enzyme activity under a certaincondition, and determining the amount on the basis of the measuredactivity value. For example, the enzyme activity can be measured byusing an appropriate amount of enzyme with a composition of the reactionmixture of 0.1 M glutamine, 0.1 M Val-Gly, and 0.1 M potassium phosphate(pH 7.6), a reaction temperature of 37° C., and a reaction time of 1 to10 minutes. For example, when the amount of the enzyme which produces 1μmol of γ-Glu-Val-Gly in 1 minute under the aforementioned conditions isdefined to be 1 U, the reaction can be performed with substrateconcentrations of 1 to 2000 mM glutamine as the γ-glutamyl group donorand 1 to 2000 mM Val-Gly, as well as an enzyme concentration of 0.1 to100 U/ml.

The reaction temperature is usually 15 to 50° C., preferably 15 to 45°C., more preferably 20 to 40° C.

Although the molar ratio of Val-Gly or a salt thereof and the γ-glutamylgroup donor in the reaction mixture may vary depending on the type ofthe γ-glutamyl group donor used for the reaction, the molar ratio ofVal-Gly:γ-glutamyl group donor is usually preferably 1:0.1 to 1:10. Theconcentrations of Val-Gly and the γ-glutamyl group donor in the reactionmixture are usually 1 to 2000 mM, preferably 100 to 2000 mM, morepreferably 100 to 1000 mM.

The amount of the mutant GGT to the substrates is usually 0.01 to 1000U, preferably 0.1 to 500 U, more preferably 0.1 to 100 U, with respectto 1 mmol of the substrates.

When a microorganism containing the mutant GGT or a processed productthereof is used, if a peptidase, especially PepD, is contained, Val-Glyas the substrate and/or γ-Glu-Val-Gly as the product may be easilydecomposed. Therefore, it is preferable to use a PepD-deficient strainas the microorganism. Alternatively, the peptidase activity can also besuppressed by adding a metal chelating agent which chelates metal ionsrequired for the enzyme activity of peptidase, such as Co²⁺, Mn²⁺, andFe²⁺, to the reaction mixture. Examples of the metal chelating agentinclude EDTA and so forth. The concentration of the metal chelatingagent in the reaction mixture is usually 0.01 to 500 mM, preferably 0.01to 100 mM, more preferably 0.1 to 10 mM. When a purified mutant GGT orpurified mutant GGT not containing the peptidase activity is used, themetal chelating agent is unnecessary, but it may be contained.

As described above, γ-Glu-Val-Gly is produced in the reaction mixture.γ-Glu-Val-Gly or a salt thereof can be collected from the reactionmixture by, for example, various chromatography techniques such as ionexchange chromatography, reversed phase high performance liquidchromatography, and affinity chromatography, crystallization andrecrystallization from a solution, and so forth.

EXAMPLES

Hereafter, the present invention will be still more specificallyexplained with reference to examples.

Example 1 Construction of GGT Expression Plasmid

A GGT expression plasmid was constructed by inserting the ggt gene ofEscherichia coli into an expression plasmid pSF12_Sm_Aet containing therpoH promoter described below.

First, in order to delete the NdeI recognition site (restriction siteoriginated in the pUC18) contained in the plasmid pSF_Sm_Aet derivedfrom pUC18 containing a peptide-producing enzyme gene derived from theSphingobacterium sp. FERM BP-8124 and the phoC promoter (WO2006/075486A1), PCR was performed by using pSF_Sm_Aet as the template and primershaving the sequences of SEQ ID NOS: 24 and 25 with “Quik ChangeSite-Directed Mutagenesis Kit” of Stratagene according to themanufacturer's protocol. The obtained PCR product was digested withDpnI, and then the Escherichia coli JM109 strain was transformed withthe reaction mixture, applied to the LB agar medium containing 100 mg/Lof ampicillin sodium (Amp), and cultured at 25° C. for 36 hours.Plasmids were extracted from the grown colonies of the transformants ina known manner, the nucleotide sequences thereof were confirmed by using3100 Genetic Analyzer (Applied Biosystems), and the plasmid having theobjective structure was designated as pSF1_Sm_Aet. The FERM BP-8124strain was designated as AJ110003, and deposited at the independentadministrative agency, National Institute of Advanced Industrial Scienceand Technology, International Patent Organism Depository (TsukubaCentral 6, 1-1, Higashi 1-Chome, Tsukuba-shi, Ibaraki-ken, 305-8566,Japan) on Jul. 22, 2002 in line with the provisions of the BudapestTreaty, and assigned with an accession number of FERM BP-8124.

Then, in order to introduce the NdeI recognition sequence intopSF1_Sm_Aet at the site of the start methionine moiety of thepeptide-producing enzyme gene derived from Sphingobacterium sp. FERMBP-8124, PCR was performed by using pSF1_Sm_Aet as the template andprimers having the sequences of SEQ ID NOS: 26 and 27 with “Quik ChangeSite-Directed Mutagenesis Kit” mentioned above. The obtained PCR productwas digested with DpnI, and then the Escherichia coli JM109 strain wastransformed with the reaction mixture, applied to the LB agar mediumcontaining 100 mg/L of Amp, and cultured at 25° C. for 24 hours.Plasmids were extracted from the grown colonies of the transformants ina known manner, the nucleotide sequences thereof were confirmed by using3100 Genetic Analyzer (Applied Biosystems), and the plasmid having theobjective structure was designated as pSF2_Sm_Aet.

Then, the phoC promoter of pSF2_Sm_Aet was replaced with the rpoHpromoter according to the following method. The rpoH promoter region wasobtained by PCR from the Escherichia coli W3110 strain genomic DNA. PCRwas performed by using the W3110 strain genomic DNA as the template, aprimer having the sequence of SEQ ID NO: 28 (rpoH promoter region havinga nucleotide sequence containing the XbaI recognition sequence at the 5′end) as the sense primer, a primer having the sequence of SEQ ID NO: 29(complementary nucleotide sequence of the rpoH promoter region having anucleotide sequence containing the NdeI recognition sequence at the 5′end) as the antisense primer, and KOD-plus- (Toyobo) as the polymerase,with 30 cycles of 94° C. for 30 seconds, 52° C. for 1 minute, and 68° C.for 30 seconds according to the manufacturer's protocol.

Then, the obtained PCR product was digested with XbaI/NdeI, andsubjected to agarose gel electrophoresis, a portion of the DNA of about0.4 kb was excised, and the DNA was ligated to the pSF2_Sm_Aet fragment(about 4.7 kb) digested with XbaI/NdeI by using DNA Ligation Kit Ver.2.1 (Takara Bio). The Escherichia coli JM109 strain was transformed withthe reaction mixture, applied to the LB agar medium containing 100 mg/Lof Amp, and cultured at 25° C. for 36 hours. Plasmids were extractedfrom the grown colonies of the transformants in a known manner, thenucleotide sequences thereof were confirmed by using 3100 GeneticAnalyzer (Applied Biosystems), and the plasmid having the objectivestructure was designated as pSF12_Sm_Aet.

The ggt gene of the Escherichia coli was obtained by PCR from theEscherichia coli W3110 strain genomic DNA. PCR was performed by usingthe W3110 strain genomic DNA as the template, a primer having thesequence of SEQ ID NO: 30 (region containing the initiation codon of theggt gene having a nucleotide sequence containing the NdeI recognitionsequence at the 5′ end) as the sense primer, a primer having thesequence of SEQ ID NO: 31 (complementary nucleotide sequence of theregion containing the initiation codon of the ggt gene having anucleotide sequence containing the PstI recognition sequence at the 5′end) as the antisense primer, and KOD-plus- (Toyobo), with 30 cycles of94° C. for 30 seconds, 52° C. for 1 minute, and 68° C. for 120 secondsaccording to the manufacturer's protocol. Then, the obtained PCR productwas digested with NdeI/PstI, and subjected to agarose gelelectrophoresis, a portion of the objective DNA of about 1.8 kb wasexcised, and the DNA was ligated to the pSF12_Sm_Aet fragment digestedwith NdeI/PstI (about 3.0 kb) by using DNA Ligation Kit Ver. 2.1 (TakaraBio). The Escherichia coli JM109 strain was transformed with thereaction mixture, applied to the LB agar medium containing 100 mg/L ofAmp, and cultured at 25° C. for 36 hours. Plasmids were extracted fromthe grown colonies of the transformants in a known manner, thenucleotide sequences thereof were confirmed by using 3100 GeneticAnalyzer (Applied Biosystems), and the plasmid having the objectivestructure was designated as pSF12_ggt.

Example 2 Preparation of pepA Gene-, pepB Gene-, pepD Gene-, pepE Gene-and pepN Gene-Disrupted Strains Derived from Escherichia coli JM 109Strain

From the Escherichia coli JM109 strain as a parent strain, PepA, PepB,PepD, PepE, and PepN non-producing strains were constructed. PepA isencoded by the pepA gene (GenBank Accession: 7439053, SEQ ID NO: 16),PepB is encoded by the pepB gene (GenBank Accession: 7437614, SEQ ID NO:18), PepD is encoded by the pepD gene (GenBank Accession: 7438954, SEQID NO: 14), PepE is encoded by the pepE gene (GenBank Accession:7438857, SEQ ID NO: 20), and PepN is encoded by the pepN gene (GenBankAccession: 7438913, SEQ ID NO: 22).

Each gene was disrupted by a method consisting of a combination of themethod called “Red-driven integration”, first developed by Datsenko andWanner (Proc. Natl. Acad. Sci. USA, vol. 97, No. 12, pp. 6640-6645,2000), and an excision system derived from λ phage (J. Bacteriol., 2002Sep., 184 (18):5200-3, Interactions between integrase and excisionase inthe phage lambda excisive nucleoprotein complex, Cho E H, Gumport R I,and Gardner J F) (refer to WO2005/010175). According to the “Red-drivenintegration” method, using a PCR product obtained by using syntheticoligonucleotides in which a part of a target gene is designed on the 5′side, and a part of antibiotic resistance gene is designed on the 3′side, respectively, as primers, a gene-disrupted strain can beconstructed in one step. By further using the excision system derivedfrom λ phage in combination, the antibiotic resistance gene incorporatedinto the gene-disrupted strain can be eliminated.

As the template for PCR, the plasmid pMW118-attL-Cm-attR was used.pMW118-attL-Cm-attR (WO2006/078039) is a plasmid obtained by insertingattL and attR genes, which are the attachment sites of λ phage, and thecat gene, which is an antibiotic resistance gene, into pMW118 (NipponGene), and the genes are inserted in the order of attL-cat-attR. PCR wasperformed by using synthetic oligonucleotides as primers havingsequences corresponding to the both ends of these attL and attR at the3′ ends and a sequence corresponding to a part of the pepA, pepB, pepD,pepE or pepN gene as the objective gene at the 5′ ends.

That is, a DNA fragment for disruption of the pepA gene was prepared byperforming PCR using pMW118-attL-Cm-attR as the template, the primershaving the sequences of SEQ ID NOS: 32 and 33, and KOD-plus- of Toyobo,with 30 cycles of 94° C. for 30 seconds, 52° C. for 1 minute, and 68° C.for 120 seconds according to the manufacturer's protocol.

The DNA fragment for disruption of the pepB gene was obtained asfollows. Namely, a fragment of about 1.0 kb locating upstream of thepepB gene was amplified by performing PCR using the Escherichia coliW3110 strain genomic DNA as the template, the primers having thesequences of SEQ ID NOS: 34 and 35, and KOD-plus- with 30 cycles of 94°C. for 30 seconds, 52° C. for 1 minute, and 68° C. for 60 secondsaccording to the manufacturer's protocol (DNA fragment A). Similarly, afragment of about 1.0 kb locating downstream of the pepB gene wasamplified by performing PCR using the primers having the sequences ofSEQ ID NOS: 36 and 37, and KOD-plus- with 30 cycles of 94° C. for 30seconds, 52° C. for 1 minute, and 68° C. for 60 seconds according to themanufacturer's protocol (DNA fragment B). Further, a fragment of about1.6 kb was amplified by performing PCR using the plasmidpMW118-attL-Cm-attR as the template, the primers having the sequences ofSEQ ID NOS: 38 and 39, and KOD-plus- with 30 cycles of 94° C. for 30seconds, 52° C. for 1 minute, and 68° C. for 120 seconds according tothe manufacturer's protocol (DNA fragment C). By using the obtained DNAfragments A, B and C in amounts of 50, 10, and 50 ng, respectively, PCRwas performed by using KOD-plus- with 10 cycles of 94° C. for 2 minutes,52° C. for 30 seconds, and 68° C. for 2 minutes according to themanufacturer's protocol. Then, second PCR was performed by using 1 μl ofeach of the obtained PCR products as the template together with theprimers having the sequences of SEQ ID NOS: 34 and 37, and KOD-plus-with 30 cycles of 94° C. for 30 seconds, 52° C. for 1 minute, and 68° C.for 4 minutes according to the manufacturer's protocol to obtain the DNAfragment for disruption of the pepB gene.

The DNA fragment for disruption of the pepD gene was prepared byperforming PCR using the primers having the sequences of SEQ ID NOS: 40and 41 and KOD-plus- with 30 cycles of 94° C. for 30 seconds, 52° C. for1 minute, and 68° C. for 120 seconds according to the manufacturer'sprotocol.

The DNA fragment for disruption of the pepE gene was prepared byperforming PCR using the primers having the sequences of SEQ ID NOS: 42and 43 and KOD-plus- with 30 cycles of 94° C. for 30 seconds, 52° C. for1 minute, and 68° C. for 120 seconds according to the manufacturer'sprotocol.

The DNA fragment for disruption of the pepN gene was prepared byperforming PCR using the primers having the sequences of SEQ ID NOS: 44and 45 and KOD-plus- with 30 cycles of 94° C. for 30 seconds, 52° C. for1 minute, and 68° C. for 120 seconds according to the manufacturer'sprotocol.

The DNA fragments for gene disruption obtained as described above wereeach purified by agarose gel electrophoresis, and introduced into theEscherichia coli JM109 strain harboring the plasmid pKD46 havingtemperature sensitive replication ability by electroporation. Theplasmid pKD46 (Proc. Natl. Acad. Sci. USA, 97:12:6640-45, 2000) includesa total 2,154 nucleotide DNA fragment of phage λ (GenBank/EMBL accessionno. J02459, nucleotide positions 31088 to 33241) containing genes codingfor the Red recombinase of the λ Red homologous recombination system (γ,β, exo genes) under the control of the arabinose-inducible P_(araB)promoter. The plasmid pKD46 is necessary for integration of the DNAfragments for gene disruption into the chromosome of the JM109 strain.Competent cells for electroporation were prepared as follows. Namely,Escherichia coli JM109 strain harboring the plasmid pKD46 was culturedat 30° C. for 20 hours in the LB medium containing 100 mg/L of Amp, andthe culture was diluted 50 times with 2 ml of the SOB medium (MolecularCloning A Laboratory Manual, 2nd edition, Sambrook, J. et al., ColdSpring Harbor Laboratory Press (1989)) containing Amp (100 mg/L). Thecells in the obtained diluted suspention were grown at 30° C. to anOD₆₀₀ of about 0.3, then added with 70 μl of 10% (v/v) L-arabinose, andcultured at 37° C. for 1 hour. Then, the obtained culture fluid wasconcentrated 65 times, and the cells were washed three times with 10%(v/v) glycerol and thereby made electrocompetent. Electroporation wasperformed by using 30 μl of the competent cells and about 100 ng of thePCR product.

After the electroporation, 0.27 mL of the SOC medium (Molecular CloningA Laboratory Manual, 2nd edition, Sambrook, J. et al., Cold SpringHarbor Laboratory Press (1989)) was added to the cell suspension, andthe cells were cultured at 37° C. for 3 hours, and then cultured at 37°C. on the LB agar medium containing chloramphenicol (Cm, 50 mg/L), andCm resistant recombinant strains were chosen. Then, in order to removethe pKD46 plasmid, the strains were cultured at 42° C. on the LB agarmedium containing Cm (50 mg/L), the obtained colonies were examined forthe Amp resistance, and the Amp sensitive strains where pKD46 had beenremoved were obtained. Disruption of the pepA gene, pepB gene, pepDgene, pepE gene, and pepN gene of the mutants identified with the Cmresistance gene was confirmed by PCR. The obtained pepA gene-, pepBgene-, pepD gene-, pepE gene-, and pepN gene-disrupted strains weredesignated as JM109ΔpepA:att-cat strain, JM109ΔpepB:att-cat strain,JM109ΔpepD:att-cat strain, JM109ΔpepE:att-cat strain, andJM109ΔpepN:att-cat strain, respectively.

Then, in order to remove the att-cat gene introduced into the pepA gene,pepB gene, pepD gene, pepE gene, and pepN gene, pMW-intxis-ts was usedas the helper plasmid. pMW-intxis-ts is a plasmid carrying a gene codingfor phage integrase (Int) and a gene coding for excisionase (Xis), andhaving temperature sensitive replication ability. If pMW-intxis-ts isintroduced into a cell, it recognizes attL or attR on the chromosome tocause recombination, thus the gene between attL and attR is excised, andonly the attB sequence remains on the chromosome. Competent cells of theJM109ΔpepA:att-cat strain, JM109ΔpepB:att-cat strain, JM109ΔpepD:att-catstrain, JM109ΔpepE:att-cat strain, and JM109ΔpepN:att-cat strainobtained above were prepared in a conventional manner, transformed withpMW-intxis-ts, and cultured at 30° C. on the LB agar medium containing100 mg/L of Amp, and Amp resistant strains were chosen. Then, in orderto remove the pMW-intxis-ts plasmid, the transformants were cultured at42° C. on the LB agar medium, and Amp resistance and Cm resistance ofthe obtained colonies were examined to obtain Cm and Amp sensitivestrains, which were strains where att-cat and pMW-intxis-ts had beenremoved, and the pepA gene, the pepB gene, the pepD gene, the pepE gene,or the pepN gene had been disrupted. These strains were designated asJM109ΔpepA strain, JM109ΔpepB strain, JM109ΔpepD strain, JM109ΔpepEstrain, and JM109ΔpepN strain, respectively.

Example 3 Construction of Mutant ggt Genes

In order to construct mutant ggt genes, PCR was performed by usingprimers corresponding to various mutant ggt genes (SEQ ID NOS: 46 to211) and pSF12_ggt mentioned in Example 1 as the template with “Quikchange Site-Directed Mutagenesis Kit” of Stratagene according to themanufacturer's protocol. The relations between the mutations and theprimers are shown in Tables 2 to 5.

After each of the obtained PCR products was digested with DpnI, theEscherichia coli JM109ΔpepA strain, JM109ΔpepB strain, JM109ΔpepDstrain, JM109ΔpepE strain, and JM109ΔpepN strain were each transformedwith the reaction mixture, applied to the LB agar medium containing 100mg/L of Amp, and cultured at 25° C. for 36 hours. Plasmids wereextracted from the grown colonies of the transformants in a knownmanner, the nucleotide sequences thereof were confirmed by using 3100Genetic Analyzer (Applied Biosystems), and the objective transformantswere used for further examination.

The plasmids introduced with various mutations were given withdesignations consisting of pSF12-ggt and indication of type of themutation. For example, a plasmid having a mutant ggt gene coding for amutant GGT having the Y444E mutation is described as pSF12-ggt(Y444E).

TABLE 2 SEQ  Introduced ID NO Sequence mutation 46CCGAACGTTGCGGGGCTGGTGG Y444A 47 CCACCAGCCCCGCAACGTTCGG 48CCGAACGTTGATGGGCTGGTGG Y444D 49 CCACCAGCCCATCAACGTTCGG 50CCGAACGTTGAAGGGCTGGTGG Y444E 51 CCACCAGCCCTTCAACGTTCGG 52CCGAACGTTAACGGGCTGGTGG Y444N 53 CCACCAGCCCGTTAACGTTCGG 54GCTTAATAACGCGATGGATGATTTC Q430A 55 GAAATCATCCATCGCGTTATTAAGC 56GCTTAATAACTGCATGGATGATTTC Q430C 57 GAAATCATCCATGCAGTTATTAAGC 58GCTTAATAACGATATGGATGATTTC Q430D 59 GAAATCATCCATATCGTTATTAAGC 60GCTTAATAACGAAATGGATGATTTC Q430E 61 GAAATCATCCATTTCGTTATTAAGC 62GCTTAATAACTTTATGGATGATTTC Q430F 63 GAAATCATCCATAAAGTTATTAAGC 64GCTTAATAACGGCATGGATGATTTC Q430G 65 GAAATCATCCATGCCGTTATTAAGC 66GCTTAATAACCATATGGATGATTTC Q430H 67 GAAATCATCCATATGGTTATTAAGC 68GCTTAATAACATTATGGATGATTTC Q430I 69 GAAATCATCCATAATGTTATTAAGC 70GCTTAATAACAAAATGGATGATTTC Q430K 71 GAAATCATCCATTTTGTTATTAAGC 72GCTTAATAACCTGATGGATGATTTC Q430L 73 GAAATCATCCATCAGGTTATTAAGC 74GCTTAATAACATGATGGATGATTTC Q430M 75 GAAATCATCCATCATGTTATTAAGC 76GCTTAATAACAACATGGATGATTTC Q430N 77 GAAATCATCCATGTTGTTATTAAGC 78GCTTAATAACCCGATGGATGATTTC Q430P 79 GAAATCATCCATCGGGTTATTAAGC 80GCTTAATAACCGCATGGATGATTTC Q430R 81 GAAATCATCCATGCGGTTATTAAGC 82GCTTAATAACAGCATGGATGATTTC Q430S 83 GAAATCATCCATGCTGTTATTAAGC 84GCTTAATAACACCATGGATGATTTC Q430T 85 GAAATCATCCATGGTGTTATTAAGC 86GCTTAATAACGTGATGGATGATTTC Q430V 87 GAAATCATCCATCACGTTATTAAGC 88GCTTAATAACTGGATGGATGATTTC Q430W 89 GAAATCATCCATCCAGTTATTAAGC 90GCTTAATAACTATATGGATGATTTC Q430Y 91 GAAATCATCCATATAGTTATTAAGC 92CCAGATGGATGCGTTCTCCGCC D433A 93 GGCGGAGAACGCATCCATCTGG

TABLE 3 SEQ  Introduced ID NO Sequence mutation 94CCAGATGGATTGCTTCTCCGCC D433C 95 GGCGGAGAAGCAATCCATCTGG 96CCAGATGGATGAATTCTCCGCC D433E 97 GGCGGAGAATTCATCCATCTGG 98CCAGATGGATTTTTTCTCCGCC D433F 99 GGCGGAGAAAAAATCCATCTGG 100CCAGATGGATGGCTTCTCCGCC D433G 101 GGCGGAGAAGCCATCCATCTGG 102CCAGATGGATCATTTCTCCGCC D433H 103 GGCGGAGAAATGATCCATCTGG 104CCAGATGGATATTTTCTCCGCC D433I 105 GGCGGAGAAAATATCCATCTGG 106CCAGATGGATAAATTCTCCGCC D433K 107 GGCGGAGAATTTATCCATCTGG 108CCAGATGGATCTGTTCTCCGCC D433L 109 GGCGGAGAACAGATCCATCTGG 110CCAGATGGATATGTTCTCCGCC D433M 111 GGCGGAGAACATATCCATCTGG 112CCAGATGGATAACTTCTCCGCC D433N 113 GGCGGAGAAGTTATCCATCTGG 114CCAGATGGATCCGTTCTCCGCC D433P 115 GGCGGAGAACGGATCCATCTGG 116CCAGATGGATCAGTTCTCCGCC D433Q 117 GGCGGAGAACTGATCCATCTGG 118CCAGATGGATCGCTTCTCCGCC D433R 119 GGCGGAGAAGCGATCCATCTGG 120CCAGATGGATAGCTTCTCCGCC D433S 121 GGCGGAGAAGCTATCCATCTGG 122CCAGATGGATACCTTCTCCGCC D433T 123 GGCGGAGAAGGTATCCATCTGG 124CCAGATGGATGTGTTCTCCGCC D433V 125 GGCGGAGAACACATCCATCTGG 126CCAGATGGATTGGTTCTCCGCC D433W 127 GGCGGAGAACCAATCCATCTGG 128CCAGATGGATTATTTCTCCGCC D433Y 129 GGCGGAGAAATAATCCATCTGG 130GCAGCCCGCGGCGAAACTGGCACG F174A 131 CGTGCCAGTTTCGCCGCGGGCTGC 132GCAGCCCGCGATTAAACTGGCACG F174I 133 CGTGCCAGTTTAATCGCGGGCTGC 134GCAGCCCGCGCTGAAACTGGCACG F174L 135 CGTGCCAGTTTCAGCGCGGGCTGC 136GCAGCCCGCGATGAAACTGGCACG F174M 137 CGTGCCAGTTTCATCGCGGGCTGC

TABLE 4 SEQ Introduced ID NO Sequence mutation 138GCAGCCCGCGGTGAAACTGGCACG F174V 139 CGTGCCAGTTTCACCGCGGGCTGC 140GCAGCCCGCGIGGAAACTGGCACG F174W 141 CGTGCCAGTTTCCACGCGGGCTGC  142GCAGCCCGCGTATAAACTGGCACG F174Y 143 CGTGCCAGTTTATACGCGGGCTGC 144GTGGTGAAAATTGGTAAAACCTG D472I 145 CAGGTTTTACCAATTTTCACCAC 146CTATAAAGGCCGCATTGCGGAAC T246R 147 GTTCCGCAATGCGGCCTTTATAG 148GATCCATATCCTGCAAATCCTCAATATTC V301L 149 GAATATTGAGGATTTGCAGGATATGGATC150 GCTGAACACCAACTTCGGTACGG T413N 151 CCGTACCGAAGTTGGTGTTCAGC 152GTAGCCCAGGCAGCAGCCGGATCATC G484S 153 GATGATCCGGCTGCTGCCTGGGCTAC 154GTGATGCCAACAGCGTCGGGCCGAAC A453S 155 GTTCGGCCCGACGCTGTTGGCATCAC 156CGTACCGAACGCGGAAGGGCTGG V443A/Y444E 157 CCAGCCCTTCCGCGTTCGGTACG 158GCTGAACACCGCGTTCGGTACGG T413A 159 CCGTACCGAACGCGGTGTTCAGC 160GCTGAACACCCATTTCGGTACGG T413H 161 CCGTACCGAAATGGGTGTTCAGC 162GTAGCCCAGGCGCGAGCCGGATCATC G484A 163 GATGATCCGGCTCGCGCCTGGGCTAC 164GTAGCCCAGGCGAAAGCCGGATCATC G484E 165 GATGATCCGGCTTTCGCCTGGGCTAC 166GGTGTGGAGAAAGATGTCTTC E38K 167 GAAGACATCTTTCTCCACACC 168CGATATGTTCGTGGATGATCAGG L127V 169 CCTGATCATCCACGAACATATCG 170GGTGGTGAATTGCATCGATTATG S498C 171 CATAATCGATGCAATTCACCACC 172GAAGCAAAAGGTCATAAAGTGGCGC Q542H 173 GCGCCACTTTATGACCTTTTGCTTC 174GGTCGAACGCAACCCGATAAGCG T276N 175 CGCTTATCGGGTTGCGTTCGACC 176CCGACCCGCGCAAAGTGGATGATTTAAC S572K 177 GTTAAATCATCCACTTTGCGCGGGTCGG

TABLE 5 SEQ Introduced ID NO Sequence mutation 178CTATACGCTGCAGACCACCTTCG N411Q 179 CGAAGGTGGTCTGCAGCGTATAG 180CCGGGCGTAGCGAACGTTGAAG P441A/Y444E 181 CTTCAACGTTCGCTACGCCCGG  182GTACCGAACGAAGAAGGGCTGG V443E/Y444E 183 CCAGCCCTTCTTCGTTCGGTAC 184GTACCGAACGGCGAAGGGCTGG V443G/Y444E 185 CCAGCCCTTCGCCGTTCGGTAC 186GTACCGAACCTGGAAGGGCTGG V443L/Y444E 187 CCAGCCCTTCCAGGTTCGGTAC  188GTACCGAACAACGAAGGGCTGG  V443N/Y444E 189 CAGCCCTTCGTTGTTCGGTAC 190GTACCGAACCAGGAAGGGCTGG V443Q/Y444E 191 CCAGCCCTTCCTGGTTCGGTAC 192GTTGAAGGGGCGGTGGGCGGTG Y444E/L446A 193 CACCGCCCACCGCCCCTTCAAC 194GGTTGGGCCGAACGGTGAGTTGTAC D561N 195 GTACAACTCACCGTTCGGCCCAACC 196GCCGCCGCGCATCCTGCGCCGCC P27H 197 GGCGGCGCAGGATGCGCGGCGGC 198GTAATCAAACTTGCCATTACTCAGTG T392C 199 CACTGAGTAATGGCAAGTTTGATTAC

Example 4 Escherichia coli Strains Deficient in each Peptidase andEvaluation of Val-Gly Degradation Ability Thereof

The JM109ΔpepA strain, JM109ΔpepB strain, JM109ΔpepD strain, JM109ΔpepEstrain, and JM109ΔpepN strain were transformed with pUC18, respectively.

Each of the obtained transformants was cultured at 25° C. for 22 hoursusing the LB medium [1.0% (w/v) peptone, 0.5% (w/v) yeast extract, and1.0% (w/v) NaCl] containing 100 mg/L of Amp. The cells in the obtainedculture fluid were washed with a 0.2 M potassium phosphate buffer (pH8.0), and a cell suspension was prepared with the same buffer. Areaction mixture containing 100 mM Val-Gly, 0.2 M potassium phosphatebuffer (pH 8.0), and the cells was prepared. The cell density was such adensity that the reaction mixture diluted 51 times showed an absorbanceof 0.2 at 610 nm. When a metal salt was added to the reaction mixture,it was added at a final concentration of 0.1 mM. Whenethylenediaminetetraacetic acid (EDTA) was added, it was added by usinga 500 mM aqueous solution thereof produced by Nakarai Tesque (pH 8.0) ata final concentration of 1 mM. The reaction conditions were 20° C. and20 hours, and Val-Gly was quantified by HPLC after completion of thereaction. The quantification conditions were as follows.

As the column, Synergi 4p Hydro-RP 80A produced by Phenomenex (particlesize: 4 microns, internal diameter: 4.6 mm, length: 250 mm) was used. Asthe eluent, Solution A (50 mM sodium dihydrogenphosphate, pH 2.5, pH wasadjusted with phosphoric acid) and Solution B (1:1 mixture of Solution Aand acetonitrile) were used. The column temperature was 40° C., and thedetection UV wavelength was 210 nm. As the gradient of the eluent, usedwere 0 to 5% Solution B for 0 to 5 minutes, 5% Solution B for 5 to 15minutes, 5 to 80% Solution B for 15 to 30 minutes, 80 to 0% Solution Bfor 30 to 30.1 minutes, and 0% Solution B for 30.1 to 50 minutes.

The results are shown in Table 6.

TABLE 6 Host bacterium strain JM109 JM109 JM109 JM109 JM109 JM109Deficient gene — ΔpepA ΔpepB ΔpepD ΔpepE ΔpepN Harbored plasmid pUC18pUC18 pUC18 pUC18 pUC18 pUC18 No addition 89.4 85.8 84.5 93.8 81.4 61.00.1 mM CoCl₂ 0.0 0.0 0.0 86.8 0.0 0.0 0.1 mM MgCl₂ 84.7 78.5 79.7 85.678.8 58.5 0.1 mM 0.0 0.0 0.0 85.7 0.0 0.0 MnSO₄ 0.1 mM NiSO₄ 80.5 71.675.5 87.3 75.1 0.0 0.1 mM ZnSO₄ 74.7 73.4 71.7 93.2 73.8 0.0 0.1 mMFeSO₄ 0.0 0.0 0.0 90.3 0.0 0.0   1 mM EDTA 94.0 91.9 91.2 91.3 90.1 90.1

As shown in Table 2, the cells of Escherichia coli wild-type straindecomposed Val-Gly in the presence of Co²⁺, Mn²⁺, or Fe²⁺ ions. Theseresults revealed that PepD mainly participates in the decomposition ofVal-Gly. In addition, when these metal ions were not added,decomposition of Val-Gly was suppressed to some extent by addition of 1mM EDTA.

Example 5 Evaluation of γ-glutamylation of Val-Gly by Mutant GGT Enzymes

The JM109ΔpepD strain was transformed with the plasmids described inExample 3. The transformants were cultured at 25° C. for 22 hours byusing the TB medium [Terrific Broth, Molecular Cloning A LaboratoryManual, 3rd edition, Sambrook, J. et al., Cold Spring Harbor LaboratoryPress (2001)] containing 100 mg/L of Amp. Each of the obtained culturefluids and an equivalent volume of a test solution (0.2 M L-glutamine,0.2 M Val-Gly, pH of the test solution was adjusted to pH 7.6 with NaGH)were mixed to start the reaction. The reaction conditions were 20° C.and 4 hours, and γ-Glu-Val-Gly was quantified by HPLC after completionof the reaction. HPLC was performed under the same conditions as thoseof Example 4.

The results are shown in Tables 7 to 9.

TABLE 7 Amino acid substitution γ-Glu-Val-Gly 1 2 3 4 (mM) 4.8 E38 K 5.8F174 I 4.8 N411 Q 43.4 T392 C 20.0 T413 N 4.2 T413 H 7.0 Q430 A 5.3 Q430C 9.6 Q430 D 11.7 Q430 E 9.9 Q430 F 8.2 Q430 G 4.7 Q430 H 9.6 Q430 I 9.0Q430 K 9.2 Q430 L 9.1 Q430 M 40.8 Q430 N 6.8 Q430 P 8.3 Q430 R 1.3 Q430S 9.5 Q430 T 4.6 Q430 V 5.4 Q430 W 3.8 Q430 Y 14.6 D433 A 10.8 D433 C0.3 D433 E 38.6 D433 F 0.1 D433 G 19.0 D433 H 0.2 D433 I 0.1 D433 K 0.1D433 L 0.1 D433 M 0.3 D433 N 5.7 D433 P 0.2 D433 Q 0.2 D433 R 0.1 D433 S5.6 D433 T 0.2 D433 V 0.1 D433 W 0.1

TABLE 8 Amino acid substitution γ-Glu-Val-Gly 1 2 3 4 (mM) D433 Y 0.2Y444 A 52.5 Y444 D 62.4 Y444 E 63.3 S572 K 25.9 G484 E 6.9 G484 S 47.9E38 K Y444 E 65.3 F174 A Y444 E 61.9 F174 I Y444 E 62.6 F174 L Y444 E64.8 F174 M Y444 E 65.2 F174 V Y444 E 66.6 F174 W Y444 E 62.8 F174 YY444 E 65.2 T246 R Y444 E 66.0 V301 L Y444 E 66.1 T392 C Y444 A 54.0T392 C Y444 E 19.9 N411 Q Q430 N 21.3 N411 Q D433 E 0.0 N411 Q S572 K40.9 T413 N Q430 N 4.1 T413 N G484 E 17.1 T413 N G484 S 22.4 T413 N S572K 4.4 T413 A Y444 E 64.0 T413 H Y444 E 70.8 T413 N Y444 E 65.4 Q430 NY444 A 53.6 Q430 N Y444 E 63.2 Q430 N Y444 D 64.7 Q430 N Y444 N 61.9D433 E Y444 A 9.3 D433 E Y444 E 8.5 P441 A Y444 E 63.1 V443 A Y444 E68.4 V443 E Y444 E 60.8 V443 G Y444 E 67.4 V443 L Y444 E 61.3 V443 NY444 E 60.8 V443 Q Y444 E 64.4 Y444 E L446 A 64.5 Y444 E A453 S 64.2Y444 E D472 I 64.1

TABLE 9 Amino acid substitution γ-Glu-Val-Gly 1 2 3 4 (mM) Y444 E G484 A65.5 Y444 E G484 S 60.1 Y444 E S498 C 66.7 Y444 E Q542 H 66.1 Y444 ED561 N 65.4 P27 H T413 N Y444 E 64.9 P27 H Y444 E G484 S 62.1 E38 K T413N Y444 E 62.6 L127 V T413 N Y444 E 62.8 T276 N T413 N Y444 E 63.1 T276 NY444 E G484 S 64.5 T413 H Y444 E G484 S 66.5 T413 N Q430 N Y444 E 65.8T413 N V443 A Y444 E 62.8 T413 N Y444 E A453 S 62.6 T413 N Y444 E S498 C62.6 T413 N Y444 E Q542 H 64.0 T413 N Y444 E G484 S 60.3 V443 A Y444 EG484 S 65.0 Y444 E G484 S Q542 H 63.4 T413 N V443 A Y444 E G484 S 62.4T413 N Y444 E A453 S G484 S 61.2 T413 N Y444 E G484 S Q542 H 61.3 T413 NY444 E G484 S S572 K 62.1 T413 N Q430 N Y444 E G484 S 69.9 T413 N Y444 EG484 E S498 C 60.8

Example 6 Evaluation of γ-glutamylation of Val-Gly by Mutant GGTEnzyme-Expressing Strains Prepared by Using Escherichia coli StrainsDeficient in Each Peptidase as Host

The Escherichia coli JM109 strain, JM109ΔpepA strain, JM109ΔpepB strain,JM109ΔpepD strain, JM109ΔpepE strain, and JM109ΔpepN strain weretransformed with pUC18, pSF12-ggt, or pSF12-ggt(Y444E) to obtaintransformants. Each of the obtained transformants was cultured at 25° C.for 20 hours using the LB medium [1.0% (w/v) peptone, 0.5% (w/v) yeastextract, and 1.0% (w/v) NaCl] containing 100 mg/L of Amp. The obtainedculture fluid was centrifuged to separate the culture fluid into wetcells and supernatant, and the wet cells were suspended in thesupernatant to prepare a cell suspension so that the suspension diluted51 times had an absorbance of 0.4 at 610 nm. This cell suspension and anequivalent volume of a test solution (0.2 M potassium phosphate buffer(pH 8.0), 0.2 M L-glutamine, 0.2 M Val-Gly) were mixed to start thereaction. The reaction conditions were 20° C. and 20 hours, andγ-Glu-Val-Gly and Val-Gly were quantified by HPLC after completion ofthe reaction under the same conditions as those of Example 4. Theresults are shown in Table 10.

TABLE 10 Val-Gly γ-Glu-Val-Gly Strain Harbored plasmid (mM) (mM) JM109pSF12-ggt 59.2 1.8 JM109 ΔpepA pSF12-ggt 62.8 1.8 JM109 ΔpepB pSF12-ggt59.6 1.9 JM109 ΔpepD pSF12-ggt 85.1 2.9 JM109 ΔpepE pSF12-ggt 59.3 1.7JM109 ΔpepN pSF12-ggt 60.7 2.1 JM109 pSF12-ggt (Y444E) 38.4 34.5 JM109ΔpepA pSF12-ggt (Y444E) 42.7 33.3 JM109 ΔpepB pSF12-ggt (Y444E) 39.431.0 JM109 ΔpepD pSF12-ggt (Y444E) 48.3 36.1 JM109 ΔpepE pSF12-ggt(Y444E) 36.7 31.2 JM109 ΔpepN pSF12-ggt (Y444E) 38.3 30.6

Explanation of Sequence Listing

SEQ ID NO: 1: Nucleotide sequence of Escherichia coli ggt geneSEQ ID NO: 2: Amino acid sequence of Escherichia coli GGTSEQ ID NO: 3: Amino acid sequence of Shigella flexneri flexneri 5 str.8401 GGTSEQ ID NO: 4: Amino acid sequence of Shigella dysenteriae Sd197 GGTSEQ ID NO: 5: Amino acid sequence of Shigella boydii strain Sb227 GGTSEQ ID NO: 6: Amino acid sequence of Salmonella Typhimurium ATCC 700720GGTSEQ ID NO: 7: Amino acid sequence of Salmonella enterica entericacholeraesuis strain SC-B67 GGTSEQ ID NO: 8: Amino acid sequence of Salmonella enterica typhi strainTy2 GGTSEQ ID NO: 9: Amino acid sequence of Klebsiella pneumoniae ATCC 202080GGTSEQ ID NO: 10: Amino acid sequence of Salmonella enterica subsp.enterica serovar A str. ATCC 9150 Paratyphi GGTSEQ ID NO: 11: Amino acid sequence of Klebsiella pneumoniae cloneKPN308894 GGTSEQ ID NO: 12: Amino acid sequence of Enterobacter cloaceae cloneEBC103795 GGTSEQ ID NO: 13: Consensus sequence of various GGT small subunitsSEQ ID NO: 14: Nucleotide sequence of Escherichia coli pepD geneSEQ ID NO: 15: Amino acid sequence of Escherichia coli PepDSEQ ID NO: 16: Nucleotide sequence of Escherichia coli pepA geneSEQ ID NO: 17: Amino acid sequence of Escherichia coli PepASEQ ID NO: 18: Nucleotide sequence of Escherichia coli pepB geneSEQ ID NO: 19: Amino acid sequence of Escherichia coli PepBSEQ ID NO: 20: Nucleotide sequence of Escherichia coli pepE geneSEQ ID NO: 21: Amino acid sequence of Escherichia coli PepESEQ ID NO: 22: Nucleotide sequence of Escherichia coli pepN geneSEQ ID NO: 23: Amino acid sequence of Escherichia coli PepNSEQ ID NOS: 24 to 31: PCR primers for preparation of pSF12_ggtSEQ ID NOS: 32 to 45: PCR primers for disruption of various peptidasegenesSEQ ID NOS: 46 to 199: PCR primers for introduction of mutation

INDUSTRIAL APPLICABILITY

The mutant GGT of the present invention has a high activity forcatalyzing γ-glutamylation of Val-Gly. Therefore, according to themethod for producing γ-Glu-Val-Gly using the mutant GGT of the presentinvention, γ-Glu-Val-Gly can be efficiently produced by using Val-Gly asa raw material.

What is claimed is:
 1. A method for producing γ-Glu-Val-Gly comprisingthe step of reacting Val-Gly with a γ-glutamyl group donor in thepresence of a γ-glutamyltransferase, a microorganism containing theenzyme, or a processed product thereof to generate γ-Glu-Val-Gly,wherein: the γ-glutamyltransferase consists of a large subunit and asmall subunit, and the small subunit has the amino acid sequence of thepositions 391 to 580 of SEQ ID NO: 2 or the amino acid sequence having ahomology of 90% or more to the foregoing amino acid sequence, and has amutation for one or more residues corresponding to one or more residuesselected from the following residues in the amino acid sequence of SEQID NO: 2: N411, T413, Q430, P441, V443, Y444, L446, A453, D472, G484,S498, Q542, D561, S572.
 2. The method according to claim 1, wherein themutation is selected from the following mutations: N411(Q) T413(H, N, A)Q430(M, N) P441A V443(E, L, G, N, Q, A) Y444(D, E, N, A) L446A A453SD472 (I) G484(S, A, E) S498C Q542H D561N S572K.
 3. The method accordingto claim 1, wherein the mutation is a mutation corresponding to any oneof the following mutations: N411Q, Q430M, Y444D, Y444E, Y444A, G484S,(T413A+Y444E), (T413H+Y444E), (T413N+Y444E), (Q430N+Y444E),(Q430N+Y444D), (Q430N+Y444N), (P441A+Y444E), (V443A+Y444E),(V443E+Y444E), (V443G+Y444E), (V443L+Y444E), (V443N+Y444E),(V443Q+Y444E), (Y444E+L446A), (Y444E+A453S), (Y444E+D472I),(Y444E+G484A), (Y444E+G484S), (Y444E+S498C), (Y444E+Q542H),(Y444E+D561N), (T413N+Y444E+V443A), (T413N+Y444E+A453S),(T413N+Y444E+S498C), (T413N+Y444E+Q542H), (G484S+Y444E+V443A),(G484S+Y444E+Q542H), (Q430N+Y444E+T413N), (T413H+Y444E+G484S),(T413N+Y444E+G484S), (T413N+Y444E+G484S+V443A),(T413N+Y444E+G484S+A453S), (T413N+Y444E+G484S+Q542H),(T413N+Y444E+G484S+S572K) (T413N+Y444E+G484S+Q430N),(T413N+Y444E+G484E+S498C).
 4. The method according to claim 1, whereinthe large subunit has the amino acid sequence of the positions 26 to 390of SEQ ID NO: 2 or the amino acid sequence having a homology of 90% ormore to the foregoing amino acid sequence.
 5. The method according toclaim 4, wherein the large subunit has a mutation corresponding to anyone of the following mutations in the amino acid sequence of SEQ ID NO:2: P27H, E38K, L127V, F174A, F1741, F174L, F174M, F174V, F174W, F174Y,T246R, T276N, V301L.
 6. The method according to claim 1, wherein thesmall subunit has the amino acid sequence of SEQ ID NO: 13 except forthe aforementioned mutation.
 7. The method according to claim 6, whereinthe small subunit has the amino acid sequence of: the positions 391 to580 of SEQ ID NO: 2, the positions 391 to 580 of SEQ ID NO: 3, thepositions 392 to 581 of SEQ ID NO: 4, the positions 388 to 577 of SEQ IDNO: 5, the positions 391 to 580 of SEQ ID NO: 6, the positions 391 to580 of SEQ ID NO: 7, the positions 391 to 580 of SEQ ID NO: 8, thepositions 400 to 589 of SEQ ID NO: 9, the positions 391 to 580 of SEQ IDNO: 10, the positions 392 to 581 of SEQ ID NO: 11, or the positions 392to 581 of SEQ ID NO: 12, or any one of these amino acid sequencesincluding substitutions, deletions, insertions, additions, or inversionsof one or several amino acid residues, except for the aforementionedmutation.
 8. The method according to claim 1, wherein the large subunithas the amino acid sequence of: the positions 26 to 390 of SEQ ID NO: 2,the positions 26 to 390 of SEQ ID NO: 3, the positions 26 to 391 of SEQID NO: 4, the positions 26 to 387 of SEQ ID NO: 5, the positions 25 to390 of SEQ ID NO: 6, the positions 25 to 390 of SEQ ID NO: 7, thepositions 25 to 390 of SEQ ID NO: 8, the positions 33 to 399 of SEQ IDNO: 9, the positions 25 to 390 of SEQ ID NO: 10, the positions 25 to 391of SEQ ID NO: 11, or the positions 25 to 391 of SEQ ID NO: 12, or anyone of these amino acid sequences including substitutions, deletions,insertions, additions, or inversions of one or several amino acidresidues, except for the mutation described in claim
 5. 9. The methodaccording to claim 1, wherein the γ-glutamyl group donor is L-glutamineor a salt thereof.
 10. The method according to claim 1, wherein theγ-glutamyltransferase, the microorganism containing the enzyme, or theprocessed product thereof is a microorganism containing the enzyme, or aprocessed product thereof, and the microorganism is a bacteriumbelonging to the family Enterobacteriaceae.
 11. The method according toclaim 10, wherein the microorganism is an Escherichia bacterium.
 12. Themethod according to claim 11, wherein the microorganism is Escherichiacoli.
 13. The method according to claim 10, wherein the microorganism isdeficient in peptidase D.
 14. The method according to claim 1, whereinthe reaction is performed in the presence of a metal chelating agent.15. A mutant γ-glutamyltransferase consisting of the following largesubunit and small subunit: (A) a large subunit which has the amino acidsequence of the positions 26 to 390 of SEQ ID NO: 2 or the amino acidsequence including substitutions, deletions, insertions, additions, orinversions of one or several amino acid residues, and able to form acomplex having the γ-glutamyltransferase activity with any one of thefollowing small subunit; (B) a small subunit which has the amino acidsequence of the positions 391 to 580 of SEQ ID NO: 2 or the amino acidsequence including substitutions, deletions, insertions, additions, orinversions of one or several amino acid residues, has any one of thefollowing mutations, and able to form a complex having theγ-glutamyltransferase activity with the above large subunit: Y444D,Y444E, (T413A+Y444E), (T413H+Y444E), (T413N+Y444E), (Q430N+Y444E),(Q430N+Y444D), (Q430N+Y444N), (P441A+Y444E), (V443A+Y444E),(V443E+Y444E), (V443G+Y444E), (V443L+Y444E), (V443N+Y444E),(V443Q+Y444E), (Y444E+L446A), (Y444E+A453S), (Y444E+D472I),(Y444E+G484A), (Y444E+G484S), (Y444E+S498C), (Y444E+Q542H),(Y444E+D561N), (T413N+V443A+Y444E), (T413N+Y444E+A453S),(T413N+Y444E+S498C), (T413N+Y444E+Q542H), (G484S+Y444E+V443A),(G484S+Y444E+Q542H), (Q430N+Y444E+T413N), (T413H+Y444E+G484S),(T413N+Y444E+G484S), (T413N+Y444E+G484S+V443A),(T413N+Y444E+G484S+A453S), (T413N+Y444E+G484S+Q542H),(T413N+Y444E+G484S+S572K), (T413N+Y444E+G484S+Q430N),(T413N+Y444E+G484E+S498C).
 16. The mutant γ-glutamyltransferaseaccording to claim 15, wherein the large subunit has any one of thefollowing mutations: P27H, E38K, L127V, F174A, F1741, F174L, F174M,F174V, F174W, F174Y, T246R, T276N, V301L.